荧光检测技术在蛋白酪氨酸激酶抑制剂高通量筛选中的应用

介绍荧光共振能量转移技术、荧光偏振免疫分析技术、荧光素酶-激酶检测技术和毛细管电泳-激光诱导荧光技术在酪氨酸激酶抑制剂高通量筛选模型中的应用情况。蛋白酪氨酸激酶与多种疾病的发生和发展密切相关,通过高通量筛选寻找其抑制剂已成为新药研发的热点之一。荧光检测技术因具有灵敏、易于检测、适于微量化和安全性高等优势,在药物的高通量筛选中被广泛使用。     

Technological Advances in High-Throughput Screening

High-throughput screening (HTS) is the process of testing a large number of diverse chemical structures against disease targets to identify 'hits'. Compared to traditional drug screening methods, HTS is characterized by its simplicity, rapidness, low cost, and high efficiency, taking the ligand-target interactions as the principle, as well as leading to a higher information harvest. As a multidisciplinary field, HTS involves an automated operation-platform, highly sensitive testing system, specific screening model (in vitro), an abundant components library, and a data acquisition and processing system. Various technologies, especially the novel technologies such as fluorescence, nuclear-magnetic resonance, affinity chromatography, surface plasmon resonance, and DNA microarray, are now available, and the screening of more than 100,000 samples per day is already possible. Fluorescence-based assays include the scintillation proximity assay, time-resolved energy transfer, fluorescence anisotropy, fluorescence correlation spectroscopy, and fluorescence fluctuation spectroscopy. Fluorescence-based techniques are likely to be among the most important detection approaches used for HTS due to their high sensitivity and amenability to automation, giving the industry-wide drive to simplify, miniaturize, and speed up assays. The application of NMR technology to HTS is another recent trend in drug research. One advantage afforded by NMR technology is that it can provide direct information on the affinity of the screening compounds and the binding location of protein. The structure-activity relationship acquired from NMR analysis can sharpen the library design, which will be very important in furnishing HTS with well-defined drug candidates. Affinity chromatography used for library screening will provide the information on the fundamental processes of drug action, such as absorption, distribution, excretion, and receptor activation; also the eluting curve can give directly the possibility of candidate drug. SPR can measure the quantity of a complex formed between two molecules in real-time without the need for fluorescent or radioisotopic labels. SPR is capable of characterizing unmodified biopharmaceuticals, studying the interaction of drug candidates with macromolecular targets, and identifying binding partners during ligand fishing experiments. DNA microarrays can be used in HTS be used to further investigate the expression of biological targets associated with human disease, which then opens new and exciting opportunities for drug discovery. Without doubt, the addition of new technologies will further increase the application of HTS in drug screening and its related fields.     

Primary cells and stem cells in drug discovery: emerging tools for high-throughput screening

Many drug discovery screening programs employ immortalized cells, recombinantly engineered to express a defined molecular target. Several technologies are now emerging that render it feasible to employ more physiologically, and clinically relevant, cell phenotypes. Consequently, numerous approaches use primary cells, which retain many functions seen in vivo, as well as endogenously expressing the target of interest. Furthermore, stem cells, of either embryonic or adult origin, as well as those derived from differentiated cells, are now finding a place in drug discovery. Collectively, these cells are expanding the utility of authentic human cells, either as screening tools or as therapeutics, as well as providing cells derived directly from patients. Nonetheless, the growing use of phenotypically relevant cells (including primary cells or stem cells) is not without technical difficulties, particularly when their envisioned use lies in high-throughput screening (HTS) protocols. In particular, the limited availability of homogeneous primary or stem cell populations for HTS mandates that novel technologies be developed to accelerate their adoption. These technologies include detection of responses with very few cells as well as protocols to generate cell lines in abundant, homogeneous populations. In parallel, the growing use of changes in cell phenotype as the assay readout is driving greater use of high-throughput imaging techniques in screening. Taken together, the greater availability of novel primary and stem cell phenotypes as well as new detection technologies is heralding a new era of cellular screening. This convergence offers unique opportunities to identify drug candidates for disorders at which few therapeutics are presently available.     

Fragment-based lead discovery: leads by design

Fragment-based lead discovery (also referred to as needles, shapes, binding elements, seed templates or scaffolds) is a new lead discovery approach in which much lower molecular weight (120-250 Da) compounds are screened relative to HTS campaigns. Fragment-based hits are typically weak inhibitors (10 microM-mM), and therefore need to be screened at higher concentration using very sensitive biophysical detection techniques such as protein crystallography and NMR as the primary screening techniques, rather than bioassays. Compared with HTS hits, these fragments are simpler, less functionalized compounds with correspondingly lower affinity. However, fragment hits typically possess high 'ligand efficiency' (binding affinity per heavy atom) and so are highly suitable for optimization into clinical candidates with good drug-like properties.     

Confocal fluorescence microscopy for high-throughput screening of G-protein coupled receptors

In the pharmaceutical industry, G-protein coupled receptors (GPCRs) are the most successful group of therapeutic targets. Finding compounds that interfere with the ligand-GPCR interaction in a specific and selective way is a major focus of pharmaceutical research today. As compound libraries of large pharmaceutical companies have increased to hundreds of thousands of test compounds, there is a growing need for miniaturization of drug discovery assays to save bioreagents and to reduce the consumption of test compounds. Due to its high sensitivity combined with a femtoliter-sized measurement volume, confocal fluorescence microscopy enables designs for GPCR binding assays with tiny sample volumes. The GPCRs are prepared in the form of plasma membrane fragments from GPCR-overexpressing cells or may be integrated into virus-like particles (VLiPs). One technique to extract binding data from confocal fluorescence experiments is the so-called fluorescence intensity distribution analysis (FIDA). In this review article, we describe the applicability of FIDA to GPCR-focussed high-throughput screening (HTS) and compare FIDA to two other GPCR-adaptable drug discovery techniques for ligand binding studies, the scintillation proximity assay (SPA) and macroscopic fluorescence polarization (FP) measurements. FIDA measures the absolute concentrations of both GPCR-bound and unbound ligand, thereby providing an internal control to the drug screening data. FIDA is amenable to work with relatively low amounts of GPCRs so that the assay may be carried out with biomembranes of a low GPCR density. Moreover, the fluorescence intensity readout of the FIDA technique may be combined with other confocal fluorescence readouts such as fluorescence anisotropy or lifetime. The combination of a low sample volume with an information-rich measurement means that confocal fluorescence spectroscopy can bring substantial benefits as a bioassay platform to pharmaceutical GPCR-directed research.     

Confocal optics microscopy for biochemical and cellular high-throughput screening

In recent years, both academia and pharmaceutical industry have produced significant advances in confocal detection and spectroscopy by laser-induced fluorescence. Confocal fluorescence studies provide information on identity, size, diffusion coefficient and concentration of the fluorescently labeled entity. This enables the establishment of sophisticated biochemical drug screening assays using the multitude of fluorescence parameters that can be observed (e.g. molecular brightness, fluorescence lifetime, anisotropy, resonance energy transfer). In cellular screening assays, confocality introduces spatial resolution in the vertical direction and reduces background fluorescence from outside the focal plane. Confocal HTS systems focusing on femtoliter-sized observation volumes allow for assay volumes far beyond current limits.     

双光子荧光影像技术在药物研发中的应用前景

双光子荧光显微成像技术具有高分辨率和高灵敏度,与激光共聚焦显微镜相比,它在成像的穿透深度上有显著提高,大大降低光毒性和光漂白,这些优点有利于双光子荧光显微成像技术用于组织样品的候选药物筛选,有望成为有效缩短新药研发周期、降低新药开发成本和提高新药命中率的手段。     

A high throughput drug screen based on fluorescence resonance energy transfer (FRET) for anticancer activity of compounds from herbal medicine

BACKGROUND AND PURPOSE: We report the development of a very efficient cell-based high throughput screening (HTS) method, which utilizes a novel bio-sensor that selectively detects apoptosis based on the fluorescence resonance energy transfer (FRET) technique. EXPERIMENTAL APPROACH: We generated a stable HeLa cell line expressing a FRET-based bio-sensor protein. When cells undergo apoptosis, they activate a protease called 'caspase-3'. Activation of this enzyme will cleave our sensor protein and cause its fluorescence emission to shift from a wavelength of 535 nm (green) to 486 nm (blue). A decrease in the green/blue emission ratio thus gives a direct indication of apoptosis. The sensor cells are grown in 96-well plates. After addition of different chemical compounds to each well, a fluorescence profile can be measured at various time-points using a fluorescent plate reader. Compounds that can trigger apoptosis are potential candidates as anti-cancer drugs. KEY RESULTS: This novel cell-based HTS method is highly effective in identifying anti-cancer compounds. It was very sensitive in detecting apoptosis induced by various known anti-cancer drugs. Further, this system detects apoptosis, but not necrosis, and is thus more useful than the conventional cell viability assays, such as those using MTT. Finally, we used this system to screen compounds, isolated from two plants used in Chinese medicine, and identified several effective compounds for inducing apoptosis. CONCLUSIONS AND IMPLICATIONS: This FRET-based HTS method is a powerful tool for identifying anti-cancer compounds and can serve as a highly efficient platform for drug discovery.     

Advances in methods for therapeutic peptide discovery, design and development

Drug discovery and development are intense, lengthy and interdisciplinary processes. Traditionally, drugs were discovered by synthesizing compounds in time-consuming multi-step experimental investigations followed by in vitro and in vivo biological screening. Promising candidates were then further studied for their pharmacokinetic properties, metabolism and potential toxicity. Today, the process of drug discovery has been revolutionized due to the advances in genomics, proteomics, and bioinformatics. Efficient technologies such as combinatorial chemistry, high throughput screening (HTS), virtual screening, de novo design and structure-based drug design contribute greatly to drug discovery. Peptides are emerging as a novel class of drugs for cancer therapy, and many efforts have been made to develop peptide-based pharmacologically active compounds. This paper presents a review of current advances and novel approaches in experimental and computational drug discovery and design. We also present a novel bioactive peptide analogue, designed using the Resonant Recognition Model (RRM), and discuss its potential use for cancer therapeutics.     

High throughput screening assay for UDP-glucuronosyltransferase 1A1 glucuronidation profiling

Development of high throughput screening (HTS) assays for evaluation of a compound's toxicity and potential for drug-drug interactions is a critical step towards production of better drug candidates and cost reduction in the drug development process. HTS assays for drug metabolism mediated by cytochrome P450s are now routinely used in compound library characterization and for computer modeling studies. However, development and application of HTS assays involving UDP-glucuronosyltransferases (UGTs) are lagging behind. Here we describe the development of a fluorescence-based HTS assay for UGT1A1 using recombinant enzyme and fluorescent substrate in the presence of an aqueous solution of PreserveX-QML (QBI Life Sciences, Madison, WI) polymeric micelles, acting as a stabilizer and a blocker of nonspecific interactions. The data include assay characteristics in 384-well plate format obtained with robotic liquid handling equipment and structures of hits (assay modifiers) obtained from the screening of a small molecule library at the University of Wisconsin HTS screening facility. The application of the assay for predicting UGT-related drug-drug interactions and building pharmacophore models, as well as the effects of polymeric micelles on the assay performance and compound promiscuity, is discussed.     

Fragment-based approach to drug lead discovery: overview and advances in various techniques

In the last 20 years the advent of new technologies, such as high-throughput screening (HTS) and combinatorial chemistry, has produced new tools for the discovery of biologically active molecules. In the past decade, fragment-based drug discovery has emerged as a more rational and focused approach that concentrates on the quality, rather than the quantity, of hits and leads. The principles behind this strategy are different from those that represented the basis of conventional HTS. The starting point of this approach is always a small chemical entity (typically MW 150-200), a fragment, with low affinity for the selected target. Fragments should satisfy key features such as diversity, reduced structural complexity, aqueous solubility and availability. Because of their small size, they occupy a smaller region of chemical space if compared with classical HTS compounds; hence, fragment libraries provide a good diversity with a relatively low number of compounds. Classical biochemical assays are often not suitable to detect the low binding affinities involved, so some well known biophysical techniques, such as nuclear magnetic resonance and x-ray, have been opportunely modified in order to render them able to perform the task. When selecting fragments suitable for subsequent optimization, a useful parameter has been introduced, the ligand efficiency, which is defined as the free energy of binding divided by the non-hydrogen atom count. Once selected, a fragment must undergo a heavy elaboration to improve binding affinity, at the same time acquiring drug-like properties. There are two main ways to go on at this point. The most common one is the so-called 'fragment evolution', consisting of a stepwise and systematic addition of chemical functionalities to the starting fragment core, together with a continuous feedback for pharmacological and physicochemical properties. The second one, less common but with great potential, is 'fragment linking': when two or more fragment hits are found to bind in adjacent regions of the target protein, they can be linked through appropriate spacers to rapidly produce a single molecule with much higher binding affinity. Two representative case histories are described: Abbott's ABT 518, an MMP (matrix metalloproteinase) inhibitor, and Eli-Lilly's LY-517717, an inhibitor of factor Xa serine protease. In addition, a list of molecules claimed to be derived from fragment approach and currently undergoing clinical trials is presented.     

Confocal fluorescence detection expanded to UV excitation: the first continuous fluorimetric assay of human steroid sulfatase in nanoliter volume

Steroid sulfatase is an enzyme that currently enjoys considerable interest as a potential drug target in the treatment of estrogen- and androgen-dependent diseases, in particular breast cancer. We have purified human steroid sulfatase to apparent homogeneity from recombinant Chinese hamster ovary cells, and we established an assay with a new fluorogenic substrate, 3,4-benzocoumarin-7-O-sulfate (1). Substrate 1 features a K(m) value of 22.5 microM, which is close to the value for the natural substrate dehydroepiandrosterone sulfate (26 microM) and much lower than the K(m) values of other synthetic substrates (276-736 microM). Importantly, the cleavage of substrate 1 can be monitored continuously during the enzymatic cleavage, since a change in fluorescence intensity is detectable at the pH where the enzyme is active; in contrast, all other synthetic substrates described so far require alkalization to reveal a measurable absorbance or fluorescence signal. The adaptation of the assay to the 96-well format allows continuous monitoring of multiple wells in a microplate fluorescence reader. Applications of the assay for the determination of IC(50) and K(i) values of novel steroid sulfatase inhibitors are presented. Most importantly the assay was transferred to the nanoscale format (1-microl assay volume) in 2080-well plates with confocal fluorescence detection. This miniaturization will permit screening with a minimum throughput of 20000 compounds per day. The system presented demonstrates that the confocal detection platform used for nanoscreening can be successfully adapted to assays for which conventional ultraviolet dyes like coumarins are necessary. This strongly broadens the application range of confocal readers in drug screening.     

Tools for GPCR drug discovery

G-protein-coupled receptors (GPCRs) mediate many important physiological functions and are considered as one of the most successful therapeutic targets for a broad spectrum of diseases. The design and implementation of high-throughput GPCR assays that allow the cost-effective screening of large compound libraries to identify novel drug candidates are critical in early drug discovery. Early functional GPCR assays depend primarily on the measurement of G-protein-mediated 2nd messenger generation. Taking advantage of the continuously deepening understanding of GPCR signal transduction, many G-protein-independent pathways are utilized to detect the activity of GPCRs, and may provide additional information on functional selectivity of candidate compounds. With the combination of automated imaging systems and label-free detection systems, such assays are now suitable for high-throughput screening (HTS). In this review, we summarize the most widely used GPCR assays and recent advances in HTS technologies for GPCR drug discovery.     

Studying biophysical barriers to DNA delivery by advanced light microscopy

Advanced light microscopy (ALM) has been intensively employed by biophysicists to reveal cellular mechanisms. As described in this review, ALM clearly has potential to enhance our understanding of the mechanisms that affect macromolecular therapeutics or nanoscopic drug vectors in biological environments. However, while in recent years confocal microscopy and related techniques became rather routinely used in drug delivery it remains challenging to extract reliable information on the biophysical behaviour of drug delivery systems from ALM measurements. This review discusses studies in which confocal imaging, fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS) and fluorescence energy transfer were employed to reveal biophysical properties of DNA and DNA containing nanoparticles in extra- and intracellular media.     

Tuberous sclerosis preclinical studies: timing of treatment, combination of a rapamycin analog (CCI-779) and interferon-gamma, and comparison of rapamycin to CCI-779

Background

Protein-protein interactions (PPIs) are challenging but attractive targets for small chemical drugs. Whole PPIs, called the 'interactome', have been emerged in several organisms, including human, based on the recent development of high-throughput screening (HTS) technologies. Individual PPIs have been targeted by small drug-like chemicals (SDCs), however, interactome data have not been fully utilized for exploring drug targets due to the lack of comprehensive methodology for utilizing these data. Here we propose an integrative in silico approach for discovering candidates for drug-targetable PPIs in interactome data.

Results

Our novel in silico screening system comprises three independent assessment procedures: i) detection of protein domains responsible for PPIs, ii) finding SDC-binding pockets on protein surfaces, and iii) evaluating similarities in the assignment of Gene Ontology (GO) terms between specific partner proteins. We discovered six candidates for drug-targetable PPIs by applying our in silico approach to original human PPI data composed of 770 binary interactions produced by our HTS yeast two-hybrid (HTS-Y2H) assays. Among them, we further examined two candidates, RXRA/NRIP1 and CDK2/CDKN1A, with respect to their biological roles, PPI network around each candidate, and tertiary structures of the interacting domains.

Conclusion

An integrative in silico approach for discovering candidates for drug-targetable PPIs was applied to original human PPIs data. The system excludes false positive interactions and selects reliable PPIs as drug targets. Its effectiveness was demonstrated by the discovery of the six promising candidate target PPIs. Inhibition or stabilization of the two interactions may have potential therapeutic effects against human diseases.     

Fluorescence spectroscopic profiling of compound libraries

Chromo/fluorophoric properties often accompany the heterocyclic scaffolds and impurities that comprise libraries used for high-throughput screening (HTS). These properties affect assay outputs obtained with optical detection, thus complicating analysis and leading to false positives and negatives. Here, we report the fluorescence profile of more than 70,000 samples across spectral regions commonly utilized in HTS. The quantitative HTS paradigm was utilized to test each sample at seven or more concentrations over a 4-log range in 1,536-well format. Raw fluorescence was compared with fluorophore standards to compute a normalized response as a function of concentration and spectral region. More than 5% of library members were brighter than the equivalent of 10 nM 4-methyl umbelliferone, a common UV-active probe. Red-shifting the spectral window by as little as 100 nm was accompanied by a dramatic decrease in autofluorescence. Native compound fluorescence, fluorescent impurities, novel fluorescent compounds, and the utilization of fluorescence profiling data are discussed.     

Formulation Development of Therapeutic Monoclonal Antibodies Using High-Throughput Fluorescence and Static Light Scattering Techniques: Role of Conformational and Colloidal Stability

In this work, we describe the application of two different high-throughput screening (HTS) techniques that can be used to determine protein stability during early formulation development. Differential scanning fluorescence (DSF) and differential static light scattering (DSLS) are used to determine the conformational and colloidal stability of therapeutic monoclonal antibodies (mAbs) during thermal denaturation in a high-throughput fashion. DSF utilizes SYPRO® Orange, a polarity-sensitive extrinsic fluorescent probe, to monitor protein unfolding. We found that melting temperatures determined by DSF have a linear correlation with melting temperatures of the first domain unfolding determined by differential scanning calorimetry, establishing DSF as a reliable method for measuring thermal stability. The DSLS method employs static light scattering to evaluate protein stability during thermal denaturation in a 384-well format. Overall comparison between mAb aggregation under typical accelerated stress conditions (40°C) and the thermal stability obtained by DSF and DSLS is also presented. Both of these HTS methods are cost effective with high-throughput capability and can be implemented in any laboratory. Combined with other emerging HTS techniques, DSF and DSLS could be powerful tools for mAb formulation optimization.     

Assay in high throughput screening

HTS (High Throughput Screening) has been put into practice in recent years. HTS is aiming to discover lead compounds for medicinal drugs. High efficiency must be achieved in all the processes including sample preparation, assay procedure, automation and data management. This review will focus on the aspects concerned with the assay technology and the efficiency in HTS. One of the major trends in HTS is assay miniaturization using high-density microplates with 384 and 1536 wells. This allows us to increase the throughput and to decrease the cost. The so-called "mix and measure" or "homogeneous" assay system, which has no separation steps such as washing or filtration, is effective for this purpose. The homogeneous assays, such as scintillation proximity assay (SPA), fluorescence energy transfer (HTRF, LANCE) and fluorescence polarization (FP), are frequently used. The reporter gene assay or the cell proliferation assay can be adapted for the homogeneous assay using high-density plates. In addition, HTS measuring the intracellular Ca2+ influx is also possible using a CCD Imager. The assay quality as well as the efficiency is also important especially in HTS. The Z'-factor provides a good tool for evaluating the quality of assays.     

Phage display for target-based antibacterial drug discovery

Increasing bacterial drug resistance and hard-to-eradicate opportunistic infections have created a need for new antibiotics. Sequencing of microbial genomes has yielded many new potential targets for antibacterial drug discovery. However, little is known about the biochemical activities of many of these targets, making it difficult to develop HTS assays for them. Peptides isolated by phage display can be used as ‘surrogate ligands’ in competition assays for screening of targets of unknown function with small-molecule libraries. These screening assays can be adapted into a variety of high-throughput formats, including those based on radioactive, luminescence or fluorescence detection.     

Stereochemistry at the forefront in the design and discovery of novel anti-tuberculosis agents

Today, 75% of new drugs introduced to the market are single enantiomers and new techniques in asymmetric synthesis and chiral separation expedites chiral drug discovery and development worldwide. The enantiomers of a chiral drug present unique chemical and pharmacological behaviors in a chiral environment, such as the human body, in which the stereochemistry of chiral drugs determines their pharmacokinetic, pharmacodynamic, and toxicological actions. Thus, it is imperative that only the pure and therapeutically active isomer be prepared and marketed. Tuberculosis (TB), a highly contagious and airborne disease that is caused by infection with Mycobacterium tuberculosis (Mtb), currently represents one of the most threatening health problems globally. The emergence of multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB), as well as HIV co-infection along with a lengthy treatment regimen, highlights an urgent need for the development of new anti-TB agents. Currently, new chiral anti-TB agents are being developed from some well-known anti-TB agents, high throughput screening (HTS) hits, and natural products. This review will focus on the reported chiral anti-TB agents together with the clinical importance of their chirality and stereochemistry.